Biological substances derived from animal cell cultivation are finding uses in a variety of medical and agricultural applications. The importance of recombinant proteins, a specific subset of biological substances, has been the basis for many new and emerging therapies and diagnostic methodologies ranging from vaccines to cancer therapies.
Cell culturing processes for the production of biological substances range in complexity from simple manually operated batch processes to complex computer controlled continuous cultivation bioreactors; for instance, from simple 50 mL spinner flasks to complex stirred-tank bioreactors of 500 L or more with automatically operated multiple measurement devices and feedback controls. The basic principle behind each process is to utilize cells as catalytic engines to produce useful biological substances such as viruses or proteins using medium in which the cells are bathed to provide both a source of required nutrients and a means of removing inhibitory waste material.
As the production of biological substances moves from the research laboratory to commercial production, competitive markets demand productivity improvements. The yield of product from each commercial bioreactor becomes critical. So to with quality, the market demands reliability and consistency of output. Current cell culturing processes readily reach their limiting conditions for production of biological substances. These limitations are imposed by the nutrient and oxygen requirements of the cells and by accumulation of inhibitory waste metabolites; and are reached well before the theoretical limits of cell growth or protein production are reached.
Not all cell types are capable of producing all biological substances. Many biological substances found in certain cells are incompatible with or even toxic to other cell types. The choice of cell types in many situations depends on the structural complexity of the end protein being produced. While protein production levels are high in prokaryotic organisms given their rapid growth and concomitant high levels of protein expression, they are not always capable of producing functional proteins as they perform no or incomplete or different post-translational and/or co-translational modifications such as glycosylation, phosphorylation and complex multi-unit macro-assembly.
Animal cells do perform the necessary complex post-translational modifications including glycosylation, phosphorylation and macro-assembly. However, some animal cells, especially mammalian cells, are difficult to grow and maintain and do not readily lend themselves to high yield production of biological substances under industrial conditions. As a subset of animal cells, insect cells are capable of glycosylation, phosphorylation and macromolecular assembly. For the production of many recombinant proteins, insect cells are an excellent choice because these cells have simple growth requirements, are highly susceptible to infection by recombinant baculoviruses engineered to produce biological substances in insect cells, and have a good safety profile.
Cell types and desired growth dynamics dictate the selection of a bioreactor type. Basic bioreactor devices include culture flasks, roller bottles, shaker flasks, stirred-tank reactors, air-lift reactors and more recently, hollow fiber reactor devices. There are advantages and disadvantages to each type of bioreactor and these advantages and disadvantages vary according to the type of cell cultured in the system and the specific properties of those cells. What works well with attached cells may not with suspended cells. Therefore, improved bioreactors need to be flexible. They should support various cell types, operate for short or long duration cultivation periods and should operate at scales ranging up to 10,000 liters.
Growth of attached cells is limited to the surface area available and when roller bottles are used, scale up of attached cell production of biological substances can demand significant amounts of space. Alternatively, for attached cells, microcarriers can be used. However, these can limit nutrient and oxygen availability to the cells and often expose them to additional sheer forces as the use of microcarriers requires a stirred tank. Additionally, matching the proper microcarrier type to the specific cell type can prove difficult.
Insect cells represent an economically important cell type with demonstrated usefulness in manufacturing biological substances. Typically, insect cells are cultured as suspensions in stirred cell bioreactors.
Unlike bacteria that are enclosed in cell walls, animal cells, and specifically insect cells, respond negatively to relatively mild hydrodynamic shear forces found in an operating bioreactor. These damaging events include bulk-fluid turbulence associated with spinner vortex formation, fluid-tank wall collisions and gas/liquid interfaces. This gas/liquid interfaces include the interface between the culture medium and head space gas with the stirred tank and between culture medium and oxygen bubbles formed during oxygen addition, such as with sparging. Insect cells are more sensitive than many other animal cells to these hydrodynamic shear forces (Wu J, King G, Daugulis A. J., Faulkner O, Bone D. H., Goosen M. F. A. (1989) Applied Microbiology and Biotechnology 32: 249). Compounding this sensitivity is the requirement of insect cells for higher oxygen levels: introduction of oxygen produces more bubbles, that is, more gas/liquid interface, and the opportunity for more hydrodynamic shear damage.
Thus, with insect cells, the mechanism for adding oxygen to the system becomes critical. First, the cells are more sensitive to the shear forces than are other animal cells. Second, more oxygen is required to grow these cells than is required to grow other animal cells. This additional oxygen requirement brings with it the probability of further cell destruction associated with increased bubbling from the higher oxygen supply and with faster stirring required to ensure even oxygen distribution. And third, when infected with baculovirus, the oxygen demand increases yet again and so too, the probability for shear related damage increases with a third factor.
Cell death is the end result of excessive shear forces, resulting from loss of membrane integrity, cell lysis, and altered metabolic activity. This insect cell sensitivity to shear forces related to high oxygen requirement is evidenced by the need for surfactant addition to the culture medium in sparged stirred tank bioreactors of any size (Murhammer D. W., Goochee C. F. (1990) Biotechnology Progress 6: 391).
During the cell culturing processes, oxygen demand increases as cell density increases. If the oxygen need is met through increased oxygen flow and stirring, shear forces increase. Thus, oxygen remains one the of key limiting factors in high density cell culture due to the need to limit shear related cell death. In turn, limiting oxygen addition restrains cell growth and makes high density culture unattainable. Furthermore, poor oxygenation directly limits output of recombinant protein with insect cell based cell culturing systems.
Thus, it would be an advance in the art to address issues that limit cell density and recombinant protein production, such as providing both a source of required nutrients and a means of removing inhibitory waste material and/or providing oxygenation that addresses the desire to reduce or limit shear related cell death from oxygenation.
Zhang et al. Biotech. Bioeng. 59(3): 351–9 (1998) relates to a high-density insect cell perfusion process utilizing an ultrasonic filter device as a means to retain cells within the bioreactor while extracting spent medium. Per cell yields of recombinant protein were similar between normal conditions (when cells were diluted to a low density and infected with a genetically engineered baculovirus) and high-density conditions, and thus failing to demonstrate, show, teach or suggest production of a recombinant protein at high cell density. And, in a perfusion system, nutrients and waste never approach equilibrium. Thus, Zhang et al. either individually or in any combination fails to teach or suggest the present invention.
Likewise, any other filters or hollow fibers or hollow fiber filter devices or uses thereof fail to teach or suggest the present invention. For instance, in contrast with certain embodiments of the present invention, filters or hollow fibers or hollow fiber filter devices can be used: by removing medium and the cells from the bioreactor vessel, passing it through the filtering device, collecting the perfused fluid containing the desired biological substance and returning the medium with its cells to the original bioreactor vessel; or as housing for cells of interest within the extra-lumenal space of a hollow fiber filter device with perfused medium passed through the capillary tubes to the cells; or by placing unencased hollow fibers directly into the fermentation tank itself so that fresh medium can be more directly provided to immobilized or attached cells.
Microbead encapsulation involves porous hollow microballoons. Culture cells attach to the internal surfaces of these porous hollow microballoons. By controlling the diameter of the microballoon and its pore sizes, relative to cell size, the thickness of the cell layers can be controlled to allow for adequate delivery of nutrients and removal of waste metabolites. Microbead encapsulation fails to teach or suggest the present invention.
Spaulding et al., U.S. Pat. No. 5,637,477, concerns a process for insect cell culture that reduces shear, in a horizontally rotating culture vessel. Spaulding et al. too, either individually or in any combination fails to teach or suggest the present invention.
Goffe, U.S. Pat. No. 5,882,918 relates to a cell culture incubator. There is no circulation of cells. Goffe, either individually or in any combination, fails to teach or suggest the present invention.
Portner et al. Appl Micro Biot. 403–414 (1998) is directed to dialysis cultures and involves a complicated dialysis process coupled with the perfusion of waste and the addition of nutrient concentrate(s) as a means to reach high cell densities wherein the removal of waste is done in a dialysis vessel connected to a semi-permeable membrane and two additional vessels (one for the addition of dialyzing fluid and the second for the removal of waste). As a result, some nutrients must also dialyze into the dialysis vessel and get wasted. Further, one or more concentrates are added directly to the culture vessel to add nutrients and support the growth of cells and to replace what is being lost in the dialysis compartment of the bioreactor.
Portner et al. state that a limitation of their design when used in a stirred tank bioreactor is oxygen limitation in their dialysis loop (p. 409). Further, in one example with mammalian cells (p. 410, hybridoma cells), Portner et al. give no data or any indication that cells actually grew to high density; and in fact, the yields of monoclonal antibodies they report after 850 hours of culture (35.4 days) were relatively low (478 mg/l or 13.8 mg/l/day). Further, Portner state in their conclusions (p. 412) that their dialysis bioreactor can be used with stationary animal cells and that for large-scale cultures of suspended cells, that an external loop can “lead to severe problems, mainly due to oxygen limitations in the loop.”
Thus, Portner et al. directly teach away from the present invention by directly teaching that a bioreactor with an external loop of circulating cells will not work. Moreover, Portner relates to the use of an open bioreactor system requiring constant addition of dialyzing fluid to a dialysis chamber and nutrient concentrates to the bioreactor. Continuous perfusion of the dialysis chamber is a variation on a perfusion system in which nutrients and waste never approach equilibrium. And, Portner et al. do not teach or suggest the addition of oxygen by in line sparging or other means, suggesting that external circulation of cells is limited by oxygen depravation.
Garnier et al., Cytotechnology 22: 53–63 (1996) relates to dissolved carbon dioxide accumulation in a large scale and high density production of TGFβ receptor with baculovirus infected Sf-9 cells: Aeration apparently involved accumulation of dissolved carbon dioxide that inhibited protein production; oxygen may serve as a carrier gas for desorbing carbon dioxide. Garnier used a low flow rate of pure oxygen with a dissolved oxygen content of 40%, and shows that there was a problem in the art, namely that higher rates of oxygen addition can result in hydrodynamic stress detrimental to the culture. Garnier fails to teach or suggest how one could provide higher rates of oxygen transfer, or to balance oxygen transfer, mechanical stress and carbon dioxide, inter alia. Garnier fails to teach or suggest the addition of oxygen by in line sparging or other means of the present invention, as well as the apparatus and methods of the present invention, inter-alia.
Karmeu et al. Biotechnology and Bioengineering 50: 36–48 (1996) is directed in on-line monitoring of respiration in recombinant-baculovirus infected and uninfected insect cell bioreactor cultures. Dissolved oxygen (DO) levels were generally at about 40%, and as to DO, the authors assert that further investigations are required to clarify the effect of DO on baculovirus-infected insect cells. Karmeu et al. may provide that respiration in insect cell cultures can be continuously monitored on-line with data from an O2 control system or an IR CO2 detector; but, fails to teach or suggest the system and apparatus of the present invention, especially the addition of oxygen by in line sparging or other means of the present invention (alone or in combination with dialyzing means), dialyzing means (alone or in combination with oxygen addition means) as in the present invention as well as other apparatus and methods of the present invention, for instance, use or adjusting of CO2 in response to pH changes inter alia (and indeed, Karmeu teaches away from such by reporting that insect cell cultures reportedly do not require HCO3−/CO2 buffering).
Nakano et al. Appl Microbiol Biotechnol 48(5): 597–601 (1997) relates to the influence of acetic acid on the growth of E. coli during high-cell density cultivation in a dialysis reactor with controlled levels of dissolved oxygen with different carbon sources (glucose and glycerol); but fails to teach or suggest methods and apparatus of the invention.
Gehin et al. Lett Appl Microbiol 23(4): 208–12 (1996) concerns studies of Clostridium cellulolyticum ATCC 35319 under dialysis and co-culture conditions. This was in batch with and without pH regulation. H2, CO2 acetate, ethanol and lactate were end-products. No synergistic action was found. Methods and apparatus of the invention are not taught or suggested by Gehin.
Schumpp et al. J Cell Sci 97(Pt4): 639–47 (1990) relates to culture conditions for high cell density proliferation of HL-60 human promyelocytic leukemia cells. While nutrient supply and metabolic end product accumulation are possible growth limiting factors, Schumpp favors a perfusion method. Accordingly, methods and appartus of the invention are not taught or suggested by Schumpp.
LaIuppa et al., “Ex vivo expansion of hematopoietic stem and progenitor cells for transplantation,” in Jane N. Winter (ed.), Blood Stem Cell Transplantation, 1997 illustrates various systems for expansion of hematopoietic stem and progenitor cells, and fails to teach or suggest methods and apparatus of the invention.
Bedard et al., Biotechnology Letters, 19(7): 629–632 (July 1997) concerns fed batch culture of Sf-9 cells which reportedly supported 3×107 cells per ml and improved baculovirus-expressed recombinant protein yields; and relates to Sf-900 II medium and nutrient additives and nutrient concentrates. While medium, additives and nutrient concentrates may be employed in the practice of the herein invention, Badard et al. fails to teach or suggest methods and apparatus of the invention. Indeed, more generally, while components and/or cells found in literature, such as herein cited literature, may be employed in the herein invention, it is believed that heretofore methods and appartus of the invention have not been taught or suggested.
Accordingly, it is believed that heretofore simple systems, e.g. closed systems, as in the present invention, where, for instance, nutrients and waste products in the bioreactor and the dialysate are in equilibrium and do not necessitate continuous perfusion (dialysis used not only for removal of waste but for addition of nutrients) and/or the issue of oxygen depletion is addressed, e.g., by the addition of oxygen directly to circulating cells, with also the issue of reducing or limiting shear related cell death due to oxygenation by reducing or limiting or eliminating shear forces from oxygen addition addressed, have not been taught or suggested. And, it is believed that heretofore, new bioreactor systems and apparatus for high-density cell growth, uses thereof, products therefrom, as described and claimed herein, as well as the herein methods for making and using such a high-density cells and products therefrom, have not been disclosed or suggested in the art.